NT Antibody Research Articles

Experimental infection of horses with West Nile virus.

A total of 12 horses of different breeds and ages were infected with West Nile virus (WNV) via the bites of infected Aedes albopictus mosquitoes. Half the horses were infected with a viral isolate from the brain of a horse (BC787), and half were infected with an isolate from crow brain (NY99-6625); both were NY99 isolates. Postinfection, uninfected female Ae. albopictus fed on eight of the infected horses. In the first trial, Nt antibody titers reached >1:320, 1:20, 1:160, and 1:80 for horses 1 to 4, respectively. In the second trial, the seven horses with subclinical infections developed Nt antibody titers >1:10 between days 7 and 11 post infection. The highest viremia level in horses fed upon by the recipient mosquitoes was approximately 460 Vero cell PFU/mL. All mosquitoes that fed upon viremic horses were negative for the virus. Horses infected with the NY99 strain of WNV develop low viremia levels of short duration; therefore, infected horses are unlikely to serve as important amplifying hosts for WNV in nature.

Tumor Necrosis Factor-α Induces Bax-Bak Interaction and Apoptosis, Which Is Inhibited by Adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.

Immunogenicity and protection in mice given inactivated influenza vaccine, MPL, QS-21 or QS-7

Background: Monophosphoryl lipid A (MPL), QS-21 and QS-7 were evaluated in mice for their ability to increase the immunogenicity and protective efficacy of formalin-inactivated (FI) influenza A/Texas/91 virus vaccine. Freund's incomplete adjuvant (FIA) was used as a positive control. Methods: Mice were inoculated twice, 28 days apart, either intramuscularly (I.M.) with vaccine mixed with phosphate buffered saline, FIA, MPL or QS21, or intranasally (I.N.) with vaccine containing QS-21 or QS-7. The mice were bled on days 0, 28 and 49 and challenged I.N. on this last day with live virus. Four days later, the lungs from each animal were assessed for influenza virus. All sera were tested for virus-specific neutralizing (Nt), hemagglutination inhibiting (HI) and ELISA antibodies. Studies to account for the mechanism(s) of adjuvant activity have been initiated. Results: FIA, MPL and QS-21 all enhanced the production of virus-specific antibodies and increased protection from pulmonary virus infection following I.M. administration. Maximal adjuvanticity occurred in groups inoculated with “low” doses of vaccine and in groups administered vaccine mixed with QS-21. Both QS adjuvants exhibited significant adjuvant activity following I.N. inoculation. Protection correlated best with levels of virus-specific serum Nt and HI antibodies. Conclusions: The present studies support continued development of adjuvants for inactivated influenza virus vaccines.

The role of apocrine released proteins in the post-testicular regulation of human sperm function.

A unifying hypothesis is presented postulating an apocrine release of several seminal proteins which mix and reaggregate in seminal fluid, thereby eventually forming particles designated either as "prostasomes", "vesiculosomes" or "seminosomes". The term "aposomes" should be restricted to the blebs released from secretory cells in the rat dorsal prostate and coagulating gland. Three different proteins present in human seminosomes along with the respective antibodies have been used to identify the localization, function and hypothetical interaction with spermatozoa. The proteins were (1) seminal vesicle-derived fibronectin, (2) prostate-derived 5'-nucleotidase and (3) a hitherto unidentified 100 kD membrane protein from epididymis, seminal vesicle and prostate. I. Fibronectin is an extracellular matrix protein which is also secreted from the seminal vesicles participating in the formation of the seminal clot. Immunofluorescence and immunoelectron microscopy revealed a relatively broad distribution pattern of fibronectin immunoreactivity on spermatozoa from different donors. Adding a fibronectin antiserum at a moderate dilution to vital spermatozoa in vitro resulted in a significant increase in sperm motility. Purified plasma fibronectin added at various concentrations to a vital sperm preparation was found to inhibit sperm motility in a dose-dependent manner. Measurement of calcium fluxes in individual sperm in the presence of fibronectin showed a significant increase. These findings point to a possible post-testicular regulatory function of seminal fibronectin. 2.5'-Nucleotidase (5'-NT) is an enzyme that hydrolyzes nucleotides such as AMP or IMP into inorganic phosphate and the respective nucleoside. The highest amount and activity of 5'-nucleotidase was present in glandular cells of the prostate; much less was detected in seminal vesicles and epididymis. On spermatozoa, the enzyme was localized on the outer leaflet of the plasma membrane covering the acrosomal region. Addition of purified enzyme to an in vitro incubation system of spermatozoa had no effect on sperm motility. A slight reduction of overall motility, however, was observed after addition of 5'-NT antibody to the spermatozoa. When 5'-nucleotidase inhibitors and adenosine channel antagonists were added to the sperm incubation system, a clear-cut inhibition of sperm motility occurred in a dose-dependent manner. This result is interpreted as indicating a significant role of ecto-5'-nucleotidase in the regulation of sperm motility. 3. A polyvalent antiserum against native human prostasomes recognized antigens in the range of 10-14 kD and of approximately 100 kD, respectively, in seminal fluid and prostate homogenates. Immunohistochemical studies revealed the presence of respective antigens in the epididymis, seminal vesicles and the prostate. Immunoelectron microscopy of ultracryo-sections showed labeling both of the apical plasma membrane in the prostate, as well as intraluminal secretory particles indicating the apocrine i.e. plasma-membrane bounded release of these particles. The secretory elements are termed "seminosomes". An affinity-purified fraction within the antiserum recognizes a 100 kD protein which is present both in the apical plasma membrane of the male genital glands, but also in the sperm head and principal piece of human spermatozoa. Incubation of spermatozoa with seminosomes and the respective purified antiserum had no effect on sperm motility. This is in contradistinction to former reports on motility increase induced by the so-called prostasomes.

Syncytium-inhibiting and neutralizing activity in maternal sera fail to prevent vertical transmission of human immunodeficiency virus type 1.

To measure the prevalence and titers of syncytium-inhibiting (SI) and neutralizing (Nt) antibodies to HIV-1 in mothers' blood close to the time of delivery and to correlate such findings with the infection status of their offspring. We analyzed serum specimens from a convenience sample of 22 HIV-infected mothers. The HIV-1 infection status of their children was determined. Forty-five percent of the women transmitted and 55% did not transmit infection to their offspring. Cord blood samples from offspring of the mothers were also studied. We measured maternal SI antibody titers against cells infected with HIV-1/SB, a strain isolated from a transmitting mother in New Haven, as well as cells infected with the more prevalent MN strain of HIV-1. We compared SI antibody titers to the SB strain in 11 matched maternal and cord blood samples. Nt antibody titers to HIV-1/SB were also measured in 20 maternal sera. Using the SB and MN strains of HIV-1, we found no difference in the prevalence or titer of SI antibody in the sera of transmitting and nontransmitting mothers. Only 35% of samples were concordant for presence or absence of SI antibody to the two strains. Furthermore the presence or absence of SI antibody in cord blood did not correlate with virus transmission. Both the frequency and titer of Nt antibody to HIV-1/SB were higher in the sera of mothers who transmitted infection when compared to those who did not. Only one-half of maternal blood samples were concordant for either the presence or absence of SI and Nt antibodies. We could not demonstrate a correlation between the presence of two types of functional antibodies (i.e. SI and Nt) to HIV-1 in the sera of pregnant women and vertical transmission. Efforts to induce or to increase such antibodies in infected mothers by immunization with vaccines or hyperimmune globulins may not alter the risk of vertical transmission.

Ockelbo virus (Togaviridae: Alphavirus) neutralizing antibodies in experimentally infected Swedish birds.

The ability of native Swedish birds to produce and maintain production of Ockelbo virus neutralizing (Nt) antibodies were evaluated experimentally between 6 June 1990 and 27 July 1991. After experimental infection of 57 birds of the orders Anseriformes and Passeriformes with Ockelbo virus, these birds were examined for Ockelbo virus Nt antibodies at 5 days, and at 1, 3, 6, 9, and 12 mo after inoculation. One month after inoculation, Nt antibodies were more prevalent in birds with a detectable viremia (100%, n = 22) than in non-viremic birds (65%, n = 26). The Nt antibody prevalence varied over time among taxa; detectable antibodies occurred earlier after inoculation and for a longer time in Anseriformes than in Passeriformes. By 5 days after inoculation, antibodies could be detected in 22 (71%) of 31 Anseriformes but in none of 16 Passeriformes. However, at 1 mo the antibody prevalences at 1 mo were similar: 84% among the Anseriformes and 73% among the Passeriformes; at 3 mo the prevalences were 50% in Anseriformes and 15% in Passeriformes. Forty-two percent of the Anseriformes had detectable antibodies even 12 mo after inoculation.

Rubella reinfection; role of neutralising antibodies and cell-mediated immunity

Background: Rubella virus may be transmitted to the fetus following rubella reinfection in early pregnancy. The mechanisms responsible for maternal-fetal transmission in women with pre-existing rubella antibodies are, however, unclear. Objectives: To evaluate the protective role of rubella specific neutralising antibodies and cell-mediated immunity in rubella reinfection. Study design: Rubella Nt antibodies were measured in sera obtained from: women who experienced rubella reinfection during pregnancy, volunteers experimentally challenged with rubella vaccine and women attending for routine rubella antibody screening. Cell-mediated immunity was assessed by measuring rubella specific LT responses among women who had experienced rubella reinfection during pregnancy. Results: Some women experienced rubella reinfection during pregnancy despite the presence of Nt antibodies in sera obtained before contact. Only 2 of 13 (15.3%) volunteers with low levels (< 15 IU/ml) of rubella antibody had detectable Nt antibodies prior to experimental challenge. Despite this, they did not develop rubella viraemia following challenge and virus excretion was detected in only one case, although the majority ( 11 13 , 84.6%) demonstrated a significant rise in antibody titre. Among pregnant women screened as having a low level of rubella antibody antenally, only 37% had detectable Nt antibodies. Among 23 women who experienced rubella reinfection during pregnancy 4 months to 6 years (mean 32 months) previously, 20 (86.9%) had a positive rubella-specific LT response. Only one of 4 cases where rubella virus was transmitted to the fetus failed to produce a a specific LT response. Conclusions: This study indicates that rubella reinfection is not associated with a lack of Nt antibodies or persistent impairment of rubella-specific LT responses.

Detection of neutralizing antibodies to Japanese encephalitis virus in the cerebrospinal fluid using a rapid microneutralization test

This paper describes the use of a rapid microneutralization test (MNT) for the detection of neutralizing (Nt) antibodies to Japanese encephalitis virus (JEV) in the cerebrospinal fluid (CSF) of Japanese encephalitis (JE) patients. In parallel, JEV specific IgM antibodies were measured using an IgM capture enzyme-linked immunosorbent assay (ELISA) (MACELISA). Nt antibodies were detected in the CSF of 156 out of 253 clinically suspected cases of JE and there was a good correlation of the results with the MACELISA. Nt antibodies were detected as early as the first day of illness. Correlation of findings with the final clinical outcome revealed that high levels of Nt antibodies (titre of ≥80) were associated with a favourable outcome. MNT using CSF proved to be a rapid, reliable diagnostic test for JE.

Measles Vaccine Immunogenicity in 6- Versus 15-Month-Old Infants Born to Mothers in the Measles Vaccine Era

The low titer of measles antibody in infants of mothers with vaccine-induced immunity may allow immunization against measles before 15 months of age. Six- and 15-month-old infants born to mothers < or = 30 years of age with no history of measles were recruited. Infants enrolled at 6 months of age were immunized with monovalent measles vaccine (Attenuvax), and maternal serum and infant pre- and postvaccination sera were obtained. Those enrolled for primary vaccination at 15 months of age received either Attenuvax (N = 12) or M-M-RII (N = 3). Six-month-old infants were revaccinated with M-M-RII at 15 months of age; pre- and postrevaccination sera were again obtained. Three antibody assays were used: a measles neutralizing assay (NT) and two enzyme immunoassays (EIA) for measles IgG and measles IgM. Among primary vaccinees, 14 of 19 infants aged 6 months (74%) developed NT antibody, as did 15 of 15 infants aged 15 months (100%). The reciprocal geometric mean titer of 6-month-old seroresponders was 23.3, significantly lower than that of the 15-month-old primary vaccinees (87.7, P < .001). Primary seroconversion rates by EIA were 53% for 6-month-old infants and 100% for those aged 15 months. Revaccination of infants who had received Attenuvax at 6 months of age resulted in 100% NT positivity; the geometric mean titer rose to equal that of the group given primary immunization at 15 months of age. Measles IgM antibody was detected in 10 of 14 infants tested 1 month after primary vaccination at 15 months, but was not detected in any of the revaccinated infants after the second dose at 15 months of age (P < .001). 1) Immunization with measles vaccine in infants born to vaccine-immune mothers at 6 months of age induced NT antibody in 74% of infants. 2) Revaccination of prior 6-month-old vaccinees at 15 months resulted in antibody titers equivalent to 15-month-old vaccinees. 3) Lack of an IgM response following revaccination suggests that even seronegative infants may be primed to respond on re-exposure to measles.

Controlled trial to evaluate protection of high-risk infants against respiratory syncytial virus disease by using standard intravenous immune globulin

We performed a randomized, controlled trial of intravenous immune globulin (respiratory syncytial virus [RSV] neutralizing [Nt] antibody titer of 1:950 in 5% solution) to evaluate protection against RSV-induced disease over two respiratory virus seasons. Forty-nine children (mean age at enrollment, 4.5 months) with severe congenital heart disease or bronchopulmonary dysplasia were randomized as follows. Twenty-four patients were followed as controls and received no immune globulin. Twenty-five patients received monthly infusions of immune globulin at a dose of 500 mg/kg of body weight. There was a similar distribution between groups of patients with heart disease and bronchopulmonary dysplasia. There were 12 culture-proven RSV infections, 6 in the prophylaxis group and 6 in the control group. There was a trend toward less severe RSV illness in immune globulin recipients, as measured by length of hospitalization. Four of the six immune globulin recipients were hospitalized for a total of 35 days (mean, 8.8 +/- 5.0 days) because of RSV illness, in contrast to 51 hospital days (mean, 12.8 +/- 7.6 days) among RSV-infected controls. We conclude that monthly infusions of standard immune globulin containing RSV Nt antibodies may be safely administered to high-risk children, but that standard intravenous immune globulin does not contain sufficient RSV Nt antibody titer to fully protect against severe RSV illness.

A 6-hour microneutralization assay for human cytomegalovirus antibody by using monoclonal antibodies

Human cytomegalovirus (HCMV) was quantified at 6, 24, and 96 h post infection by counting the number of infected cells which were stained by the immunoperoxidase technique using monoclonal antibodies reactive to different HCMV non-structural and structural proteins. A linear dose-effect relationship was found at the three indicated times. Three rapid neutralization (Nt) assays (one for each time) were developed, using the same virus inoculum. Human sera from individuals with primary, reactivated or remote HCMV infection gave comparable Nt antibody titers with the three assays. The 6-hour rapid Nt assay appears highly suitable for determination of HCMV immune status and provides results the same day of blood collection.

Antibodies to 5'-nucleotidase (CD73), a glycosyl-phosphatidylinositol-anchored protein, cause human peripheral blood T cells to proliferate.

Human peripheral blood T cells were stimulated to proliferate when cultured with submitogenic doses of PMA and goat antibodies to 5'-nucleotidase (5'-NT). The degree of proliferation, as measured by [3H]TdR incorporation on day 3, was similar to that achieved by stimulation with PHA. Anti-5'-NT antibodies had no effect on PHA-induced proliferation. Maximal stimulation was achieved with 0.6 to 1.0 ng/ml of PMA and 125 micrograms/ml of IgG isolated from a goat anti-5'-NT antiserum. Both intact IgG and F(ab')2 fragments were stimulatory. IL-2R expression and IL-2 secretion were also induced by anti-5'-NT antibodies and PMA. Anti-5'-NT-induced proliferation was inhibited greater than 95% by a murine anti-IL-2 receptor mAb and required less than 0.3% monocytes. Similar results have been obtained with a murine mAb specific for 5'-NT. As expected, anti-5'-NT antibodies and PMA did not induce the proliferation of ecto-5'-NT-T cells isolated by cell sorting. Pretreatment of total T cells with phosphatidylinositol-specific phospholipase C removed an average of 89% of the 5'-NT activity from the cell surface and also inhibited by 83% the ability of the cells to proliferate in response to anti-5'-NT antibodies and PMA. Thus, the activation signal provided by anti-5'-NT antibodies is apparently transduced, in large part, by a form of the enzyme that is attached to the membrane via glycosyl-phosphatidylinositol linkage. These data suggest that 5'-NT may play a role in lymphocyte activation as has been proposed for other glycosyl-phosphatidylinositol-anchored lymphocyte surface proteins.

Functional characterization of ecto-5'-nucleotidase-positive and -negative human T lymphocytes.

Functional studies were performed on human peripheral blood T lymphocytes stained with goat anti-5'-nucleotidase antibodies and separated into ecto-5'-nucleotidase (ecto-5'-NT)-positive and -negative populations using the FACSTAR fluorescence-activated cell sorter. On the average, ecto-5'-NT+ T cells contained 34 +/- 13% CD4+ and 55 +/- 15% CD8+ cells, whereas ecto-5'-NT-T cells contained 65 +/- 12% CD4+ and 23 +/- 8% CD8+ cells. Staining with anti-5'-NT antibodies did not significantly alter the ability of unseparated T cells to proliferate in response to PHA or PMA, or in a MLR. However, prior incubation with anti-5'-NT antibodies did inhibit the ability of irradiated T cells to provide help for PWM-stimulated Ig synthesis by as much as 55%. In five separate experiments, ecto-5'-NT-T cells demonstrated an equal or better ability to incorporate [3H]TdR after PHA stimulation or in a MLR, as compared with ecto-5'-NT+ T cells. Similarly, ecto-5'-NT- T cells were not diminished in their ability to provide help for autologous B cells in a PWM-driven system. Clearly, the inability of ecto-5'-NT- T cells from patients with a variety of immunodeficiency diseases to function in these assays cannot be explained solely by their lack of ecto-5'-NT activity. In contrast, ecto-5'-NT-positive and -negative T cells showed markedly different dose-response curves for proliferation in response to PMA. Ecto-5'-NT+ T cells responded to lower doses of PMA (1.0 ng/ml) than did ecto-5'-NT- T cells and showed a two- to eight-fold greater rate of [3H]TdR incorporation at 3 to 10 ng of PMA per ml. Ecto-5'-NT+ T cells may have a protein kinase C that is more accessible or more easily activated or may utilize an alternate pathway of activation when stimulated with low concentrations of PMA.

Synthesis of immunoglobulin G by pokeweed mitogen- or Epstein-Barr virus-stimulated human B cells in vitro is restricted to the ecto-5'-nucleotidase positive subset.

Ecto-5'-nucleotidase (ecto-5'-NT) is believed to be a maturation marker for human B lymphocytes because its expression increases during normal development and is reduced in many patients with B cell immunodeficiencies. To determine whether this enzyme defines functional subsets of B lymphocytes, human peripheral blood B cells, separated into ecto-5'-NT positive and negative populations by using goat anti-5'-NT antibodies and the fluorescence-activated cell sorter, were compared for their ability to secrete polyclonal immunoglobulin. Both populations synthesized equivalent quantities of IgM in response to a T cell-dependent (PWM) or T cell-independent (EBV) stimulator of polyclonal immunoglobulin biosynthesis. However, ecto-5'-NT+ B lymphocytes synthesized 8- to 26-fold more IgG per cell than ecto-5'-NT- B cells. These data provide the first direct evidence that ecto-5'-NT is a marker for the functional maturation of human B cells and support the hypothesis that ecto-5'-NT deficiency in patients with hypogammaglobulinemia results from a block in B lymphocyte maturation.

161 ANTI-5′-HUCLEOTIDASE ANTIBODIES CAUSE HUMAN PERIPHERAL BLOOD T CELLS TO PROLIFERATE

Human peripheral blood T cells were stimulated to proliferate when cultured with subraitogenic doses of phorbol myristate acetate (PMA) and goat antibodies to ecto-5′-nucleotidase (ecto-5′-NT). The degree of proliferation, as measured by 3H-thymidine incorporation on day 3, was similar to that achieved by stimulation with phytohemagglutinin (PHA). Anti-5′-NT antibodies had no effect on PHA-induced proliferation. Maximal stimulation was achieved with 0.6-1.0 ng PMA/ml and 150 μg/ml of IgG isolated from a goat anti-5′-NT antiserum. Both intact IgG and F(ab')2 fragments were stimulatory. IL-2 receptor expression and IL-2 secretion were also induced by anti-5′-NT antibodies and PMA. As expected, anti-5′-NT antibodies and PMA did not induce the proliferation of ecto-5′-NT− T cells isolated by cell sorting. Pretreatment of total T cells with phosphatidylinositol (PI)-specific phospholipase C removed 80% of the ecto-5′-NT activity from the cell surface and also inhibited the ability of the cells to proliferate in response to anti-5′-NT antibodies and PMA by 84%. Thus, the activation signal provided by anti-ecto-5′-NT antibodies is apparently transduced by a form of the enzyme which is attached to the membrane via PI-linkage. These data suggest that ecto-5′-NT may play a role in lymphocyte activation as has been proposed for other PI-linked lymphocyte surface proteins including Thy-l, T cell activating protein, TAP, and the rat alloantigen RT-6.

160 FUNCTIONAL CHARACTERIZATION OF ECTO-5′-NUCLEOTIDASE (ECTO-5′-NT) POSITIVE AND NEGATIVE HUMAN LYMPHOCYTES

Human T and B lymphocytes, separated into ecto-5′-NT positive and negative populations with goat antl-5′-NT antibodies and the fluorescence-activated cell sorter, were characterized in functional assays. Ecto-5′-NT− T cells proliferated as well as, or better than, ecto-5′-NT+ T cells after stimulation with phytohemagglutinin or in a mixed lymphocyte reaction. Ecto-5′-NT− T cells also provided more help for pokeweed mitogen (PWM)-stimulated immunoglobulin synthesis than ecto-5′-NT+ T cells. Therefore, the inability of ecto-5′-NT deficient lymphocytes from patients with immunodeficiency diseases to respond in these assays cannot be attributed solely to their reduced ecto-5′-NT activity. In contrast, ecto-5′-NT+ T cells proliferated in response to ten-fold lower doses of phorbol esters than ecto-5′-NT+ T cells, suggesting that ecto-5′-NT+ T cells may utilize unique biochemical pathways of cellular activation or may have a preferential ability to respond to certain stimuli. Ecto-5′-NT positive and negative B cells were tested for the ability to synthesize IgM and IgG after stimulation with PWM and Epstein Barr virus. Although both groups of cells synthesized IgM, the synthesis of IgG was restricted to the ecto-5′-NT+ sub-population. These data provide the first direct evidence that ecto-5′-NT is a marker for the functional maturation of human B cells and demonstrate that ecto-5′-NT is different from other human B cell surface antigens such as IgD, Leu 8, and HB-4 which are lost as B cells mature.

Distribution of ecto-5'-nucleotidase on subsets of human T and B lymphocytes as detected by indirect immunofluorescence using goat antibodies.

Human T and B lymphocyte subsets were characterized for ecto-5'-nucleotidase (ecto-5'-NT) expression by two-color immunofluorescence by using polyclonal goat antibodies to 5'-NT and murine monoclonal antibodies to T and B cell subsets. Anti-5'-NT antibodies were prepared by immunizing a goat with purified human placental 5'-NT. Lymphocyte surface 5'-NT was detected with F(ab')2 fragments of immune goat IgG followed by biotinylated F(ab')2 rabbit anti-goat IgG and fluorescein isothiocyanate-avidin. Lymphocyte cell surface antigens were detected with phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD16, and anti-CD19. HB-4, an antigen present on a major subset of human peripheral blood B cells, was detected with murine monoclonal anti-HB-4 and PE-anti-mouse-kappa. Analysis showed that ecto-5'-NT was expressed on 32 +/- 7% of CD3+, 19 +/- 6% of CD4+, and 50 +/- 21% of CD8+ T cells, but not on CD16+ lymphocytes. Ecto-5'-NT was also expressed on 81 +/- 8% of adult peripheral blood B cells as defined by PE-anti-CD19; HB-4 was expressed on 84 +/- 7% of CD19+ cells. The two populations of B cells were not identical, however, because HB-4 was co-expressed on only 79 +/- 18% of ecto-5'-NT+ B cells. Two-color immunofluorescent staining of T cells from a patient with congenital agammaglobulinemia and low T cell ecto-5'-NT activity revealed reduced percentages of ecto-5'-NT+ cells in his CD3+, CD4+, and CD8+ populations. Thus, reduced ecto-5'-NT activity by enzyme assay was paralleled by reduced numbers of 5'-NT molecules on the cell surface. Two-color immunofluorescent staining of B cells from a patient with hypogammaglobulinemia and low B cell ecto-5'-NT activity also revealed markedly reduced expression of 5'-NT. HB-4 expression was normal, however, suggesting that the patient's B cells were blocked in maturation subsequent to the acquisition of HB-4 but prior to that of ecto-5'-NT. These results demonstrate that anti-5'-NT antibodies will be valuable tools for analyzing ecto-5'-NT expression and lymphocyte maturation in patients with immuno-deficiency diseases.

Congenital Guinea Pig Infection with Attenuated Junin Virus Strains

Guinea pigs born from mothers infected before or during pregnancy with 10(3) PFU of the attenuated XJC13 or XJ0 strains of Junin virus (JV) by the intramuscular route showed 31.5% mortality that was not attributable to the mothers' clinical condition or to lack of care. There was a slight drop in mortality rate when the mothers were infected at the beginning or end of their gestation period. JV isolation from the 9 offspring killed from 1 to 125 days of age proved that virus transmitted transplacentally or soon after birth was able to persist, although titers were not higher than 10(2.7) PFU/g of tissue in various organs, including brain. Cell-associated viremia could thus account for viral spread after birth. Since an active humoral response was detected in the same animals, although Nt antibody titers were below 1:16, a state of tolerance did not exist in these congenitally infected animals. The carrier state appeared to modify guinea pig susceptibility to JV; after challenge with the pathogenic XJ strain of JV, 2 animals survived and developed normal humoral responses, while half of the remaining animals did not show typical signs of Argentine hemorrhagic fever. Although JV persistence appeared to cause no deleterious effects in surviving guinea pigs, its long-term risk remains to be determined.

Neutralization Test by New Isolates of Human Rotavirus

AbstractThre newly isolated human rotaviruses in MA104 cells were used as antigens in the neutralization test of paired sera of infants and young children with acute gastroenteritis during three epidemic seasons. Twenty‐three of 41 paired sera (56%) from outpatients with acute gastroenteritis responded with development of NT antibody to one, two, or three antigens. Of 23 paired sera which showed significant rises of antibody to any strain, 20 paired sera (87%) responded to strain Hochi, 15 cases (65%) to strain Ito, and 16 cases (70%) to strain Nemoto. Ten of 14 cases that were seronegative to any isolated strain at onset of illness showed significant rises of antibodies to three strains. Three strains were determined to be subgroup 2 by RNA pattern and were recently found to be similar if not identical to serotype 3 and 4 by Wyatt et al. The neutralization test showed extensive immunologic cross‐reativity among these strains. The rate of acquisition of serum antibody by age was similar with the three antigens.

Immunological characterisation of rubella virion polypeptides.

Antibodies specific for rubella virion polypeptide, VPI, secreted by clones of hybridoma cells or produced in rabbits in response to specific antigenic stimulation, located determinants for haemagglutination inhibiting (HI) and neutralising (Nt) antibodies on this envelope component.